Chemical composition and anti-inflammatory activity of copaiba oils from Copaifera cearensis Huber ex Ducke, Copaifera reticulata Ducke and Copaifera multijuga Hayne--a comparative study.

Copaiba oil is an oleoresin obtained from the Copaifera L. genus (Leguminoseae) commonly featured in anti-inflammatory recipe prescribed by Amazonian traditional medical practitioners and featured in Europe and North America pharmacopeias of the past. Chemical and anti-inflammatory activity investigations from the copaiba oils obtained from Copaifera multijuga Hayne, Copaifera cearensis Huber ex Ducke and Copaifera reticulata Ducke species have proved that, although similar, these oleoresins possess varied composition and anti-inflammatory activity. Chromatographic studies showed that the main compound among sesquiterpenes was beta-caryophyllene (57.5, 19.7 and 40.9%, respectively), followed by alpha-humulene, alpha-copaene, alpha-bergamotene, delta-cadinene, with different amounts in each oleoresin. Among the diterpenes, copalic acid was the main component from Copaifera multijuga Hayne (6.2%) and was found in all the oleoresins studied. In Copaifera cearensis Huber ex Ducke, clorechinic (11.3%) and hardwickiic acids (6.2%) were the major diterpenes while kaurenoic (3.9%) and kolavenic acids (3.4%) predominated in Copaifera reticulata Ducke. The pharmacologic effects of the three oleoresins were evaluated in vitro by measuring the NO production by murine macrophages and in vivo using the zymosan induced pleurisy model in mice. The Copaiba Oil from Copaifera multijuga Hayne (100 mg/kg) was the most potent, inhibiting both NO production and the pleurisy induced by zymosan. The oleoresins from Copaifera cearensis Huber ex Ducke and Copaifera reticulata Ducke were also able to inhibit NO production and the pleurisy but with less intensity.

Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium.

The resins and leaves of species of Protium are commonly used by folk medicine. In the present study, we analyse the pharmacological effects of essential oils obtained by steam distillation (leaves and resin) from Protium species. Analysis by gas chromatography (GC) coupled to mass spectrometry and retention indices calculations demonstrate that the resin oil is constituted mainly of monoterpenes and phenylpropanoids: alpha-terpinolene (22%), p-cymene (11%), p-cimen-8-ol (11%), limonene (5%) and dillapiol (16%), whereas sesquiterpenes predominate as the volatile constituents of the leaves. The resin of Protium heptaphyllum (PHP) and leaves of P. strumosum (PS), P. grandifolium (PG), P. lewellyni (PL) and P. hebetatum (PHT) were screened for anti-inflammatory activity by the use of mouse pleurisy model induced by zymosan (500 microg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity), for antinociceptive effect (by means of preventing mice abdominal writhings), as well as NO production from stimulated macrophages and proliferation of neoplasic cell lines: Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma) and J774 (mouse monocytic cell line). The oils from PHP, PS and PL were able to inhibit protein extravasation but no sample inhibited total or differential leucocyte counts after administrating p.o. (100 mg/kg) 1 h before stimulation with zymosan. The oils from PG, PL and PHT inhibited neutrophil accumulation whereas PHP and specially PL inhibited LPS-induced eosinophil accumulation in mouse pleural cavity. PHT was also able to inhibit mononuclear cells accumulation. Antinociceptive effect was not observed, when animals received oral administration of the essential oils (100 mg/kg). In vitro treatment with essential oils (100 microg/well) changed the NO production from stimulated mouse macrophages. PHP inhibited in 74% and PS in 46% the LPS-induced NO production. In contrast, treatment with PL was able to increase in 49% the NO production. Cell lines proliferation was affected by the oils assayed in the range of 60-100% for Neuro-2a, 65-95% for SP2/0 and 70-90% for J774. Taken together these results showed that essential oils could be useful as efficient pharmacological tools.

Opposite cellular accumulation and nitric oxide production in vivo after pleural immunization with M. leprae or M. bovis BCG.

Mycobacteria as intracellular pathogens have evolved mechanisms to survive within macrophages. Our previous data showed that M. leprae (ML), unlike M. bovis BCG, did not induce an inflammatory response in the mice subcutaneous tissue. Further, ML inhibited BCG-induced foot pad oedema and seemed to transform macrophages in epithelioid cells. Since these mycobacteria share common antigens, here we seeked to compare the acute and chronic cellular response evoked by ML and BCG in pleurisy of a mycobacteria-susceptible mice (BALB/c). The total leukocytes, the cell type that migrated to the pleural cavity and macrophage activation assayed by nitric oxide release were determined. Live or dead BCG Moreau recruited the same extent of cells, essentially monocytes and neutrophils, dose-dependently, in both acute and chronic pleurisy. BCG-induced eosinophilia was observed only in the acute response (after 24 h of injection). A significant nitric oxide release by pleural macrophages was triggered by BCG Moreau without previous activation. Nevertheless, ML failed to recruit leukocytes to the pleural space or to lead to nitric oxide production despite the number of bacilli used and the time studied (1, 7 or 14 days after injection). Although these mycobacteria have common antigens that cross-react, these data show a distinct ability of ML or BCG to recruit cells to the pleural space and to activate pleural macrophage for nitric oxide production in vivo.

The role of gammadelta T lymphocytes in lipopolysaccharide-induced eosinophil accumulation into the mouse pleural cavity.

LPS induces an accumulation of eosinophils in the pleural cavity that requires resident macrophages and lymphocytes, but is independent of IL-5 production. In the present study we investigated the involvement of different T lymphocyte subsets on the modulation of LPS-induced eosinophil accumulation into the pleural cavity of mice. Within 4 h after LPS injection the number of neutrophils in the pleural cavity increased significantly. Mononuclear cell counts increased after 12 h, while a significant rise on eosinophil counts was observed only after 24 h. T lymphocytes counts were increased in the pleural cavity 24 and 48 h after LPS administration. This T lymphocyte accumulation was accounted for by an influx of the gammadelta+ subset, while CD4+ and CD8+ subsets did not accumulate in the pleural cavity after LPS stimulation. All those changes had resolved 96 h after LPS injection. Depletion of T lymphocytes by treatment with mAb anti-Thy 1.0 inhibited the eosinophil accumulation triggered by LPS. Aiming to clarify which T lymphocyte subset would be involved in the LPS-induced eosinophil accumulation, we depleted mice of various T lymphocyte subpopulations using specific Abs. Depletion of either CD4+ or CD8+ subsets failed to inhibit LPS-induced eosinophil migration. In contrast, when mice were treated with anti-gammadelta+ T lymphocyte mAb, a significant reduction of LPS-induced eosinophil accumulation was observed. Similarly, the administration of LPS in BALB/c-nu/nu mice induced the expected significant influx of eosinophils into the pleural cavity. Our results indicate that the gammadelta+ T lymphocytes are centrally involved in LPS-induced eosinophil accumulation in mice.